Antigen-specific IgG detection

ABSTRACT

The invention concerns a method for the determination of antigen-specific antibodies of the immunoglobulin G class in the presence of immunoglobulins of the M class in body fluids by incubation with at least two different receptors R 1  and R 2  and optionally additional receptors, an essential component of R 2  being a binding partner in monomeric form, a reagent for determining an antigen-specific antibody of the immunoglobulin G class as well as the use of binding partners in monomeric form for the determination of an antigen-specific antibody of the immunoglobulin G class.

The invention concerns a method for the determination ofantigen-specific antibodies of the immunoglobulin G class in body fluidsby incubating the sample with at least two different receptors R₁ and R₂wherein both receptors are capable of binding specifically to theantibody, R₁ is bound or can be bound to a solid phase and R₂ carries alabel, separating the solid from the liquid phase and measuring thelabel wherein a conjugate of a binding partner in monomeric form whichis specifically recognized by the antibody to be determined and a labelis used as R₂.

In particular the invention concerns a method for the specific detectionof immunoglobulins of the IgG class in the presence of immunoglobulinsof the IgM class.

In response to the introduction of foreign substances the immune systemof a mammalian organism produces antibodies which are also calledimmunoglobulins. They defend against foreign substances which are alsoreferred to as antigens. The immunoglobulins can be divided into fivedifferent classes. One distinguishes between immunoglobulins of the M,G, A, E and D classes. Each of these five immunoglobulin classes differin the composition of the heavy chain which is referred to as the μ, γ,α, ε and δ chain.

Each immunoglobulin class has a different function in the organism.Immunoglobulins of the M class appear with the first contact with theantigen the so-called first immunization. However, the concentration ofthese immunoglobulins decreases rapidly as the infection progresses. Theimmunoglobulins of the G class are firstly slowly formed after a firstimmunization and occur in large quantities when there is a secondinfection with the same antigen. The immunoglobulins of the A class arefound on the mucous membrane surfaces of the organism and areresponsible for the defence processes there. The immunoglobulins of theE class are mainly responsible for allergic reactions. The exactfunction of immunoglobulins of the D class is hitherto unknown.

The individual immunoglobulin classes occur in very differentconcentrations in the blood. Thus immunoglobulins of the G class (IgG)are the major class in normal human serum with a share of about 75% thatcorresponds to a serum content of 8 to 18 mg/ml. The second mostfrequently occurring immunoglobulin is IgA which has an average serumconcentration of 0.9 to 4.5 mg/ml. Immunoglobulins of the M class arepresent at a concentration of 0.6 to 2.8 mg/ml, immunoglobulins of the Dclass are present at a concentration of 0.003 to 0.4 mg/ml. Theproportion of IgE antibodies is lowest which only occur at aconcentration of 0.02 to 0.05 μg/ml in serum.

For the differential diagnosis of many diseases it is important todetect antibodies of one or several quite particular immunoglobulinclasses which are specific for a particular antigen. A satisfactorydiagnosis of viral, bacterial and parasitic infections can only beensured by means of a class-specific antibody test or by excluding thepresence of certain immunoglobulin classes (e.g. detection of IgG andIgA antibodies but no detection of IgM antibodies). This is particularlyimportant for the differentiation between fresh or acute infections andinfections that have occurred earlier as well as for the clinicalmonitoring of the course of an infection. The class-specific detectionof antibodies is especially important for HIV, hepatitis A, hepatitis B,toxoplasmosis, rubella and chlamydia infections. The class-specificdetection of antibodies specific for a particular antigen is alsonecessary for the determination of the titre of protecting antibodiesand to check the success of an immunization. Hence from a diagnosticview point there is great interest in the detection especially ofantibodies of the non-acute stages of infections such IgG and IgAantibodies.

Various methods have been described in the state of the art fordetecting antibodies of a particular class that are specific for anantigen. Hence antigen-specific antibodies of a particular class arefrequently detected by binding the specific antibodies to a solid phasecoated with the specific antigen. The immunoglobulins (Ig) specific forthe antigen which are now bound to the solid phase are detected bybinding antibodies which are specifically directed towards human Ig of acertain class to the Ig molecules to be detected. The antibodiesdirected towards human Ig are provided with a label by means of whichthe detection takes place. However, such a test procedure is onlypossible if all unspecific non-bound Ig is removed by washing before thereaction with the class-specific labelled antibodies directed towardshuman Ig. Thus a one-step test procedure as is often required forautomated systems is not possible.

According to the method described in the U.S. Pat. No. 4,292,403antigen-specific antibodies of a particular immunoglobulin class aredetected by immobilizing a class-specific antibody on a solid phasewhich binds sample antibodies to be determined, subsequently adding thespecific antigen and binding the bound antigen to a further labelledantibody that is specific for the antigen. However, a disadvantage ofthis method is that all antibodies of the class to be determined mustbind to the class-specific immobilized antibody. The sample antibodiesare not bound antigen-specifically. This could impair the sensitivity ofthe test since there may not be sufficient free binding sites for theantigen-specific antibody. Several wash steps are also necessary in thistest procedure. This method does not enable a one-step test procedure.

One possibility of carrying out an antibody detection in a one-step testis provided by the so-called bridge test. The bridge test concept isdescribed in EP-A-0 280 211. In this method a first receptor which iscapable of specific binding to the antibody to be determined is bound toa solid phase such as for example an antigen. The antibody to bedetermined binds to the solid phase-bound antigen. In addition a furtherspecific antigen is present in the test mixture which is provided with alabel. The antibody is detected by means of the label. In this test allantigen-specific antibodies are detected and not only the antibodies ofa particular class.

In EP-A-0 307 149 and in the U.S. Pat. No. 5,254,458 methods based onthe bridge test principle are disclosed for the detection of antibodieswhich are directed specifically towards an antigen. In this casepeptides produced recombinantly which are derived from a certain epitopeof the antigen are used to bind the antibody to be detected. A peptideis immobilized to a solid phase. The sample antibody binds to thepeptide. A further labelled peptide is bound to the sample antibody forthe detection. The recombinant peptides are expressed in differentorganisms in order to increase the specificity of the test. Also in thismethod antibodies of all classes bind to the peptides. It is notpossible to for example differentiate between IgG and IgM antibodies.

EP-A-0 386 713 describes a method for the detection of antibodiesagainst HIV using two solid carriers in which different HIV antigens areimmobilized on both solid carriers which are each contacted with analiquot of a sample and a labelled HIV antigen wherein the presence ofantibodies is detected by a positive reaction in at least one of thetests. Polypeptides produced recombinantly are disclosed as HIVantigens. A method based on Western blot is disclosed in EP-A-0 627 625in which HIV antibodies can also be detected by means of recombinantproteins or synthetic peptides. However, both methods do not allow theclass-specific detection of antigen-specific antibodies.

The previous methods do not enable an antigen-specific antibody of acertain immunoglobulin class to be detected in a one-step method. Theimmunological methods of detection known from the state of the art basedon the bridge test concept in which a labelled antigen and an antigencapable of binding to a solid phase are used, do indeed enable aone-step test. However, up to now it has only been possible to jointlydetect antibodies of the IgG and IgM classes using this simpleprinciple.

Therefore the object was to provide an improved method for the detectionof antibodies of a non-acute infection directed towards a specificantigen i.e. in particular of the IgG class. At the same time the methodshould ensure that IgM antibodies of the same specificity are notdetected. This method should preferably consist of a one-step testprinciple in order to be used advantageously in automated systems.

This object is achieved by the method according to the invention for thedetermination of an antigen-specific antibody of the immunoglobulin Gclass by incubating the sample with at least two different receptors R₁and R₂ wherein both receptors are capable of specifically binding to theantibody, R₁ is bound or can be bound to a solid phase and R₂ carries alabel which is characterized in that a conjugate of a binding partner inmonomeric form that is specifically recognized by the antibody to bedetermined and a label is used as R₂.

The method according to the invention allows the determination ofantigen-specific antibodies of the immunoglobulin G class in samples inwhich antibodies of the IgM class of the same antigen-specificity arepresent.

The IgA, IgD and IgE antibodies present in the sample which have thesame specificity as the IgG antibodies to be detected occur in muchlower concentrations than the IgG antibodies. In particular the IgD andIgE classes are present in concentrations that are several orders ofmagnitude below the IgG concentration so that their reactivity in thedetection method does not or hardly changes the measured result. IgAantibodies are present at concentrations that correspond to about 10% ofthe total IgG content. Hence IgA antibodies would also presumably bedetermined in this method. Since the main purpose of the method is todetect antibodies of the non-acute infection, the joint detection of IgGand IgA antibodies which are both antibodies of the non-acute infectionis not critical. Since IgG antibodies are the major immunoglobulin classin the non-acute infection the term IgG detection is used in thefollowing. It is essential that IgM antibodies of the same antigenspecificity which only occur in large quantities in an acute infectionmust not be detected.

It has surprisingly turned out that the use according to the inventionof binding partners in a monomeric form in a bridge test based on thedouble-antigen test principle enables antibodies of the IgG class to beexclusively detected which are specifically directed towards aparticular antigen. Antibodies of the IgM class of the same specificitywhich are present in the same sample surprisingly do not react or onlyto a negligibly weak extent with monomeric peptides and thus do notinterfere with the IgG detection. The term “negligibly weak” means thatthe antigen binding sites of the IgM antibodies are not detectably boundby the binding partners in monomeric form. This is presumably due to themuch lower affinity of the pentameric IgM antibodies for monomericepitopes compared to the IgG antibodies present in the form ofindividual molecules.

Hence a successive test procedure for separating the IgM antibodies isnot absolutely necessary in the method according to the invention sincethese do not interfere. A particular advantage of the method istherefore the simplicity of the test procedure.

Apart from the so-called wet tests in which the test reagents arepresent in a liquid phase, all standard dry test formats which aresuitable for the detection of proteins or antibodies can also be used.In these dry tests or test strips as described for example in EP-A-0 186799, the test components are applied to a carrier. Hence if the methodaccording to the invention is carried out in a test strip format no washstep is necessary. However, the method according to the invention ispreferably carried out as a wet test.

It is possible to incubate all receptors and the sample together and tocarry out the method in one step. This optionally requires only one washstep after the incubation.

Normally two different receptors R₁ and R₂ are used to carry out themethod according to the invention. If a wet test is used, the receptorR₂ is present in a liquid phase. R₁ can be present in a liquid phase oralready bound to the solid phase. If R₁ and R₂ are present in a liquidphase, they are preferably at the same concentration. Concentrationratios of R₁:R₂ of 0.5:1.0 to 1.0:5.0 have proven to be well suited. Theoptimal concentration ratios can easily be tested out by a personskilled in the art.

If a receptor capable of binding to a solid phase but which is not yetbound to the solid phase is used as R₁, the sample is then incubatedwith the receptors R₁ and R₂. In this process the sample antibody bindsto R₁ and R₂. This incubation can occur in the presence of the solidphase. A complex is formed in this process comprising solidphase-R₁-sample-antibody-R₂.

Subsequently the solid phase is separated from the liquid phase, thesolid phase is optionally washed and the label of R₂ is measured. Thelabel is usually measured in the solid phase it can, however, also bedetermined in the liquid phase.

If the incubation of the sample with R₁ and R₂ is carried out in theabsence of the solid phase, then the entire test mixture mustsubsequently be contacted with the solid phase, the washing optionallycarried out and the label measured.

If the receptor R₁ is already in a solid phase-bound form, then thesample and receptor R₂ are added to the solid phase-bound receptor R₁and incubated together. The further procedure corresponds to the processstated above.

It is, however, also possible to carry out the method according to theinvention in several steps. In this case it is expedient to firstlyincubate the sample with the receptors R₁ and R₂. The complex of R₁, R₂and antibody to be determined that is formed is subsequently incubatedwith other receptors whereby this can be carried out in several steps.The further test procedure corresponds to the previously describedmethod.

The receptor R₂ is a conjugate of a binding partner in monomeric formand a label. The binding partners according to the invention inmonomeric form contain exactly one epitope region or only one bindingsite for the antibody to be determined i.e. a structure that reactsimmunologically specifically with the IgG antibody to be determined. Themonomeric structure of the binding partner is important to ensure thatonly the antigen-specific IgG antibodies to be detected bind to thebinding partner in monomeric form and not the interfering IgM antibodiesof the same specificity.

The epitope region can for example be derived from an antigen or ananti-idiotype antibody. The epitope regions can, in the case of thebinding partners in monomeric form, also be derived from sugar and/orlipid structures or combined structures with peptide, lipid and/or sugarcomponents. All structures that can be derived from an epitope regioncan be used which have a binding site to which the antibody of the IgGclass to be detected specifically binds in the presence of IgMantibodies of the same specificity. The only prerequisite for thebinding site i.e. for the binding partners used in monomeric form, isthat the specific capability of binding to IgG is retained. Thiscondition also applies to the case where sugar or lipid structures arepresent in the binding site.

According to the invention it is also possible to use binding partnersin monomeric form which flank or overlap with the binding site to whichthe IgG antibody to be detected specifically binds. Hence it is alsopossible to detect cross-reacting IgG antibodies whose binding siteoverlaps with the epitope to be detected. Therefore a mixture of bindingpartners in monomeric form is preferably used to detect theantigen-specific IgG antibodies.

Peptides are preferably used as binding partners in monomeric form. Inthe case of a protein as an analyte a binding site is understood as apeptide, the sequence of which is part of the protein sequence of aprotein antigen and to which an antibody directed towards this protein,which in the case of the present invention is an IgG antibody,specifically binds. In addition to these peptides a binding site is alsounderstood to include peptides with amino acid sequences which have anessentially equivalent specificity and/or affinity of binding to the IgGantibody to be detected as the aforementioned peptides. These peptidescan preferably be derived from the aforementioned peptides bysubstitution, deletion or insertion of individual amino acid residues.

All peptides can be used which have a binding site to which the IgGclass antibody to be determined bind specifically even in the presenceof IgM antibodies of the same specificity. The only prerequisite for thebinding site i.e. for the peptide used, is that its ability tospecifically bind to IgG is retained. A binding site is understood as apeptide whose sequence is part of the protein sequence of a proteinantigen (analyte) and to which an antibody directed against this proteinspecifically binds which in the present invention is an IgG antibody. Inaddition to these peptides a binding site is also understood to includepeptides with amino acid sequences which have an essentially equivalentspecificity and/or affinity of binding to the IgG antibody to bedetermined as the aforementioned peptides. These peptides can preferablybe derived from the aforementioned peptides by substitution, deletion orinsertion of individual amino acid residues.

Peptides according to the invention which correspond to a specificbinding site are also understood to include peptide derivatives in whichone or several amino acids have been derivatized by a chemical reaction.Examples of peptide derivatives according to the invention are inparticular those molecules in which the backbone or/and reactive aminoacid side groups, for example free amino groups, free carboxyl groupsor/and free hydroxyl groups, have been derivatized. Specific examples ofderivatives of amino groups are sulfonamides or carboxamides,thiourethane derivatives and ammonium salts for example hydrochlorides.Carboxyl group derivatives are salts, esters and amides. Examples ofhydroxyl group derivatives are O-acyl or O-alkyl derivatives. Thepeptides are preferably produced by chemical synthesis according tomethods known to a person skilled in the art and do not need to beespecially elucidated here. In principle the peptides can also beproduced by means of recombinant methods. However, longer polypeptidesoften tend to dimerize or polymerize so that the peptides are preferablyproduced by chemical synthesis in order to ensure monomeric properties.

In addition the term peptide derivative also encompasses such peptidesin which one or several amino acids are replaced by naturally occurringor non-naturally occurring amino acid homologues of the 20 “standard”amino acids. Examples of such homologues are 4-hydroxyproline,5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, β-alanine and4-aminobutyric acid. The peptide derivatives must have an essentiallyequivalent specificity or/and affinity of binding to the IgG antibodiesto be determined as the peptides from which they are derived.

Peptides according to the invention which correspond to a specificbinding site are also referred to as peptide-mimetic substances namedpeptide-mimetics in the following which have an essentially equivalentspecificity or/and affinity of binding to the IgG antibodies to bedetermined as the aforementioned peptides or peptide derivatives.Peptide-mimetics are compounds which can replace peptides with regard totheir interaction with the antibody to be determined and can have ahigher stability than the native peptides in particular towardsproteinases and peptidases. Methods for the production ofpeptide-mimetics are described in Giannis and Kolter, “Angew. Chem.” 105(1993), 1303-1326 and Lee et al., Bull. Chem. Soc. Jpn. 66 (1993),2006-2010.

The length of a binding site i.e. the length of a monomeric peptideaccording to the invention is usually at least 4 amino acids. The lengthis preferably between 4 and 20, 6 and 15 or particularly preferably 9and 12 amino acids. In the case of peptide-mimetics or peptidederivatives an analogous length is necessary with regard to the size ofthe molecule.

The monomeric peptides according to the invention as a binding partnerin a monomeric form contain the epitope to which the IgG antibody to bedetermined binds specifically. However, further flanking peptidesequences which no longer correspond to the specific epitope may bepresent at the N-terminal and/or at the C-terminal end of the peptide.Furthermore it is possible that the peptide is provided with spacergroups familiar to a person skilled in the art. The only prerequisitesare that the peptide as a binding partner in a monomeric form isactually present as a monomer and the ability to bind to the IgGantibodies to be determined is retained.

A further component of the receptor R₂ is the label. A directlydetectable substance is preferably used as a label for example achemiluminescent, fluorescent or radioactive substance or a metal sol,latex or gold particle. Enzymes or other biological molecules are alsopreferred as the label such as for example haptens. Digoxigenin is aparticularly preferred label among the haptens. Processes for labellingare familiar to a person skilled in the art and do not need to beelucidated further here. The label is detected directly in a well-knownmanner by measuring the chemiluminescent, fluorescent or radioactivesubstance or the metal sol, latex or gold particle or by measuring thesubstrate converted by the enzyme.

The label can also be detected indirectly. In this case a furtherreceptor which itself is in turn coupled to a signal-generating groupbinds specifically to the label of R₂ such as a hapten such asdigoxigenin. The signal-generating group for example a chemiluminescent,fluorescent or radioactive substance or an enzyme or gold particle isdetected by methods familiar to a person skilled in the art. An antibodyor an antibody fragment can for example be used as the further receptorwhich binds specifically to the label of R₂. If this indirect detectionof the label is used then the R₂ label is preferably digoxigenin oranother hapten and the detection is carried out via an antibody coupledto peroxidase which is directed towards digoxigenin or towards thehapten.

An essential component of the receptor R₁ is a binding partner which iscapable of specific binding to the IgG antibody to be determined. Thereceptor R₁ can be directly bound to the solid phase or capable ofbinding to the solid phase. A binding partner in a monomeric form as inreceptor R₂ can be used as the binding partner which is capable ofspecific binding to the IgG antibody to be determined. However, it isalso possible to use binding partners which are not present in amonomeric form i.e. the binding partner can have more than one epitopeor binding site. It is important that the ability of the binding partnerto specifically bind to the IgG antibodies to be determined is retained.However, binding partners in a monomeric form and particularlypreferably peptides are also used for the receptor R₁. The peptidescontained in R₁ are produced by the same methods as the peptides for R₂.

The antibody-specific binding partners or peptides contained in thereceptors R₁ and R₂ may be identical or different, but both must becapable of simultaneously binding to the IgG antibody to be determined.

R₁ can either be bound directly to the solid phase. The direct bindingof R₁ to the solid phase is achieved by methods known to a personskilled in the art. R₁ can also be indirectly bound to the solid phaseby means of a specific binding system. In this case R₁ is a conjugatewhich is composed of a peptide as elucidated above and a reactionpartner of a specific binding system. A specific binding system is inthis case understood as two partners which can react specificallytogether. In this case the binding capability can be based on animmunological reaction or on another specific reaction. A combination ofbiotin and avidin or biotin and streptavidin is preferably used as aspecific binding system. Other preferred combinations are biotin andantibiotin, hapten and anti-hapten, Fc fragment of an antibody andantibody against this Fc fragment or carbohydrate and lectin. One of thereaction partners of this specifically bindable pair is then a part ofthe conjugate that forms the receptor R₁.

The other reaction partner of the specific binding system is thenpresent in a solid phase. The other reaction partner of the specificbinding system can be bound to an insoluble carrier material byconventional methods known to a person skilled in the art. In this casea covalent as well as an adsorptive binding is suitable. Solid phasesthat are particularly suitable are test tubes or microtitre plates madeof polystyrene or similar plastics the inner surfaces of which arecoated with the reaction partner of the specific binding system.Particulate substances such as latex particles, molecular sievematerials, glass beads, plastic tubes etc. are also suitable andparticularly preferred. Porous layered carriers such as paper can alsobe used as the carrier.

In a preferred embodiment of the method according to the invention aconjugate composed of a binding partner in a monomeric form and areaction partner of the specific binding system is used as R₁. In thispreferred embodiment the receptors R₁ and R₂ as well as the sample whichcontains the IgG antibody to be determined are incubated together. Inthis process the peptide components of the receptors R₁ and R₂ reactspecifically with the IgG antibodies to be determined. This complexcomposed of R₁, sample antibody and R₂ is bound to the solid phase whichis coated with the other reaction partner of the specific binding systemby means of the reaction partner of the specific binding system which isa component of R₁. As a result the entire complex composed of R₁, sampleantibody and R₂ is bound to the solid phase. After the solid phase hasbeen separated from the liquid phase and optionally washing the solidphase, the label of R₂ is detected by methods known to a person skilledin the art. This test procedure enables IgG antibodies to be detectedspecifically in the presence of IgM antibodies of the same specificity.

In a further preferred embodiment of the method according to theinvention an additional receptor is used in addition to the receptors R₁and R₂. In this test procedure a conjugate composed of a binding partnerin monomeric form and a reaction partner of a specific binding systemsuch as for example biotin is used as R₁. For this the receptors R₁ andR₂ as well as the sample which contains the IgG antibody to bedetermined are incubated together. In this process the peptidecomponents of the receptors R₁ and R₂ react specifically with the IgGantibodies to be determined. Binding to the solid phase which is coatedwith the other reaction partner of the specific binding system (forexample with streptavidin) is achieved by means of the one reactionpartner of a specific binding system that is a component of R₁. As aresult the entire complex composed of R₁, R₂ and sample antibody isbound to the solid phase. After separating the solid phase from theliquid phase and optionally washing the solid phase, the complex boundto the solid phase is incubated with an additional receptor whichspecifically recognizes the label of R₂. The further receptor is coupledto a signal-generating group such as an enzyme. After a further optionalwashing step, the sample antibody is detected via the signal-generatinggroup, in this case by the substrate converted by the enzyme. If thistest procedure is used digoxigenin is preferably used as the R₂ label.The additional receptor in this case is composed of an antibody orantibody fragment directed towards digoxigenin and the enzymeperoxidase. In this test procedure the incubation of the sample with R₁and R₂ and the additional receptor can also be carried out concurrently.

This test procedure is also very well suited for an application toautomated systems but requires two or several washing steps. A majoradvantage of this test procedure becomes apparent if it is intended todetect several antigen-specific antibodies such as e.g. HIV antibodiesagainst gp41, p17 etc. In such a case the additional receptor can beused as a universal label since this additional receptor specificallyrecognizes the R₂ label.

All biological liquids known to a person skilled in the art can be usedas samples. Body fluids such as whole blood, blood serum, blood plasma,urine, saliva etc. are preferably used as the sample.

In addition to the sample, the solid phase and the aforementionedreceptors, other additives which may be required depending on theapplication such as buffer, salts, detergents, protein additives such asBSA may be present in the test mixtures. The necessary additives areknown to a person skilled in the art or can be determined by him in asimple manner.

In order to ensure that IgM antibodies or rheumatoid factors do notinterfere with the antigen-specific IgG detection, it is possible tooptionally use additional measures for interference reduction. These forexample include the use of reducing substances such as dithiothreitol(DTT), dithioerythritol (DTE) or β-mercaptoethanol in the approachdisclosed in EP-B-0 341 439. In addition anti-Fd antibodies canoptionally be used to eliminate interference by rheumatoid factors. Sucha concept is disclosed in WO 96/14338. The various measures for reducinginterference can be used individually or in any combination.

A further subject matter of the invention is a reagent for thedetermination of an antigen-specific antibody of the immunoglobulin Gclass which, in addition to the usual test additives for immunoassayssuch as buffers, salts, detergents etc., contains a receptor R₂ capableof binding to the antibody to be determined which is composed of abinding partner in a monomeric form and a label.

A subject matter of the invention is also a reagent for thedetermination of an antigen-specific antibody of the immunoglobulin Gclass which, in addition to the usual test additives for immunoassays,contains two receptors R₁ and R₂ capable of binding to the antibody tobe determined of which R₁ is capable of binding to a solid phase and R₂carries a label wherein an essential component of receptor R₂ is abinding partner in a monomeric form.

A further subject matter of the invention is also a reagent for thedetermination of an antigen-specific antibody of the immunoglobulin Gclass which, in addition to the usual test additives for immunoassays,contains two receptors R₁ and R₂ capable of binding to the antibody tobe determined of which R₁ is capable of binding to a solid phase and R₂carries a label wherein an essential component of both receptors is abinding partner in a monomeric form.

Furthermore a subject matter of the present invention is the use ofbinding partners in a monomeric form to determine an antigen-specificantibody of the immunoglobulin G class.

The invention is elucidated by the following examples.

EXAMPLES

1. Reactivity with <HIV 2>MABs (IgG and IgM) when using Monomeric andMultimeric Epitopes

Description of the Test Procedure

Biotin-labelled and digoxigenin-labelled monomeric (test A) ormultimeric (test B) antigens (HIV 2) react with sample antibodies and astreptavidin-coated solid phase (incubation at 25° C. or 37° C., ca. 60to 180 min, in this example: 120 min, 25° C.). After a wash step theimmune complex bound to the wall reacts with ananti-digoxigenin-peroxidase conjugate (incubation at 25° C. or 37° C.,ca. 30 to 120 min in this example: 60 min 25° C.). After a further washstep the peroxidase conjugate-labelled immune complex is detected by asubstrate reaction (conjugate incubation 60 min at 25° C.). In generalthe conjugate incubation can be carried out at 25° C. or 37° C., for ca.30 to 120 min.

The reaction steps (apart from the substrate reaction) take place in aTris/HCl buffer (pH 7.5, 50 to 150 mM in this example 100 mM) containingca. 0.05 to 0.4% detergent (here 0.2% polidocanol) and ca. 0.5%protein/protein derivative additives (here peptone from lactalbumin andBSA among others).

In this case the sample antibodies are monoclonal mouse antibodies (IgMand IgG) against a HIV 2 epitope diluted to ca. 2-20 μg/ml in anti-HIVnegative human serum.

TABLE 1 Test results comparison of tests A and B: Absorbances in mA TestA Test B (monomeric (multimeric Samples epitopes) Evaluation epitopes)Evaluation MAB < HIV2 > 2764 positive 2800 positive IgG 20.3.1 MAB <HIV2 > 9999 positive 3974 positive IgG 23.5.3 MAB < HIV2 > 6 negative3271 positive IgM 2.6.6 MAB < HIV2 > 6 negative 4250 positive IgM 2.11.7MAB < HIV2 > 6 negative 6442 positive IgM 2.22.8

The use of HIV2-specific binding partners in a monomeric form enablesthe specific detection of IgG antibodies against HIV2. Antibodies of theIgM class of the same antigen specificity are not recognized (test A).

When HIV2-specific binding partners in a multimeric form are used it isnot possible to discriminate between IgG and IgM (test B).

2. Reactivity with Serum Antibodies of a HIV2 Seroconversion of aChimpanzee

Experimental procedure as example 1.

TABLE 2 Test results of a chimpanzee seroconversion with tests A and B:Absorbances in mA Sequential serum samples Test A Test B after(monomeric (multimeric infection epitopes) Evaluation epitopes)Evaluation week 0 (on 6 negative 98 negative infection) status: noantibodies week 1 after 7 negative 89 negative infection status: noantibodies week 3 after 6 negative 673 positive infection status: IgMantibodies week 7 after 250 borderline 589 positive infection status:IgM and IgG antibodies week 10 after 8767 positive 6845 positiveinfection status: IgG antibodies

The use of binding partners in a monomeric form enables theseroconversion after a HIV2-infection to be detected. The advantage oftest A is particularly apparent in week 3 after the infection: only IgMantibodies are present which are not detected by the binding partners ina monomeric form. Test A only shows a positive signal after IgGantibodies appear. Test B which uses multimeric epitopes is not able todistinguish between IgG and IgM of the same specificity.

3. Reactivity with Serum Antibodies of a HIV1 Seroconversion of aPatient

Experimental procedure as example 1, but antigens of HIV 1.

TABLE 3 Test results of a HIV1 seroconversion with tests A and BAdsorbances in mA Sequential serum samples after the Test A Test B first(monomeric (multimeric examination epitopes) Evaluation epitopes)Evaluation day 0 30 negative 89 negative status: no antibodies day 14 28negative 78 negative status: no antibodies day 26 35 negative 9999positive status: IgG antibodies day 35 250 borderline 9999 positivestatus: IgM and IgG antibodies day 40 589 positive 9151 positive status:IgM and IgG antibodies

Test A enables a reliable detection of IgG antibodies also in the caseof an infection with HIV1 (analogous to HIV2 according to example 2).

4. Detection of IgG Against Rubella with Peptides that Contain MonomericEpitopes

Description of the Test Procedure

Rubella-specific IgG is detected by means of the binding partnersaccording to the invention in a monomeric form on an Elecsys® 2010instrument from Boehringer Mannheim GmbH, Germany according to themanufacturer's instructions. The following reagents are used:

Reagent R1: cyclic peptide of rubella E1 antigen, biotinylated. Trisbuffer, pH 7.5, 0.2% Myrij, 0.2% BSA, 0.1% R-IgG

Reagent R1: cyclic peptide of rubella E1 antigen, ruthenylated. Trisbuffer, pH 7.5, 0.2% Myrij, 0.2% BSA, 0.1% R-IgG.

Human serum samples are used as samples.

The test is carried out in the following steps:

1. 30 μl sample+65 μl R₁+65 μl R₂

2. incubation at 37° C., 9 min

3. addition of 40 μl SA-coated magnetic beads

4. incubation at 37° C., 9 min

5. detection reaction: measuring the electrochemiluminescent signal

TABLE 4 Results Samples Characterization counts IU/ml No. 1 IgM neg.,IgG neg. 1359 0.5 No. 3 IgM neg., IgG neg. 1370 0.5 No. 8 IgM neg., IgGneg. 1338 0.5 No. 1.3 IgM pos., IgG neg. 929 0.1 No. 1.4 IgM pos., IgGneg. 981 0.2 No. 255 IgM neg., IgG pos. 11256 83 No. 272 IgM neg., IgGpos. 26254 192 No. 278 IgM neg., IgG pos. 4453 32 No. 283 IgM neg., IgGpos. 49328 363

Only samples which contain antigen-specific IgG are recognized aspositive. Samples which only contain antigen-specific IgM (No. 1.3 and1.4) are not recognized according to the invention as positive.

What is claimed is:
 1. A method for the determination of anantigen-specific antibody of the immunoglobulin G class in a biologicalsample comprising the steps of: a. forming a mixture by combining saidsample with at least two different receptors, R₁ and R₂, in which eachof said receptors binds specifically to said antibody via itsantigen-binding site according to the double antigen test principle, R₁is bound to a solid phase and R₂ comprises a conjugate of a bindingpartner in monomeric form and a label, thereby forming a complexcomprising solid phase-R₁-antibody-R₂-label, characterized in that thesample is combined together simultaneously with R₁ and R₂ in one step,b. separating the solid phase complex from the mixture, and c. measuringsaid label bound to said antibody via R₂ as a measure of said antibodypresent in said sample.
 2. The method of claim 1, wherein R₁ comprises abinding partner in monomeric form.
 3. The method of claim 2, wherein thebinding partner in monomeric form comprising R₁ is present in an amountgreater than or the same as the amount of binding partner in monomericform comprising R₂.
 4. The method of claim 1, wherein R₁ is bound tosaid solid phase by a specific binding system selected from the groupconsisting of biotin/avidin, biotin/streptavidin, biotin/antibiotin,hapten/antihapten, Fc fragment of an antibody/antibody against said Fcfragment, and carbohydrate/lectin.
 5. The method of claim 1, whereinsaid label is selected from the group consisting of chemiluminescent,fluorescent and radioactive substances, enzymes and biologicalmolecules.
 6. The method of claim 1, wherein said mixture includes anadditional receptor which specifically binds to said label, wherein saidadditional receptor comprises a conjugate of a receptor specific forsaid label and a second label, wherein said second label indirectlybound to said antibody is determined as a measure of said antibodypresent in said sample.
 7. The method of claim 1, wherein said antibodyis an HIV 2 antibody.
 8. The method of claim 1, wherein said antibody isan HIV 1 antibody.
 9. The method of claim 1, wherein said antibody is arubella antibody.